Cell division, which includes the process of nuclear division (mitosis) and the process of cytoplasmic division (cytokinesis), is initiated at the molecular level by a cascade of protein phosphorylations. The kinases catalyzing the phosphorylation events therefore mediate the many morphological changes that accompany mitosis. During cell division, the chromosomes condense, the nuclear envelope dissolves, the cell detaches from neighboring cells and the extracellular matrix, and the cytoskeleton rearranges to organize the movements necessary to segregate the chromosomes and ultimately divide the cell into two. Because faithful replication and division of the genetic material is crucial to propagation of physiologically functional cells, considerable attention has been devoted to the characterization of proteins exhibiting kinase enzymatic activity during mitosis. A number of disease states and/or disorders may result from cell division errors arising from either aberrant activation or functional mutations in the molecular components of the kinase cascades. For example, cell division errors result in hyperproliferative disorders, such as cancer.
A mutation in a Drosophila serine/threonine kinase, designated aurora, was found to be responsible for a chromosomal segregation error arising from defective spindle-pole behavior. Several Aurora-like kinases have since been identified in a number of organisms. A single Aurora named Ipl1 (Increase-in-ploidy 1) was identified in Saccharomyces cerevisiae in a genetic screen for mutants that were defective in chromosome segregation. Schizosaccharomyces pombe also has a single Aurora called Aurora-related kinase (Ark1). Aurora is thus the name given to a family of serine-threonine protein kinases that regulate many processes during cell division. In general, aurora kinases are involved in the control of the centrosome and nuclear cycles, and appear to function in mitotic processes including chromosome condensation, spindle dynamics, kinetochore-microtuble interactions, chromosome orientation, and establishment of the metaphase plate. They are also involved in cytokinesis. Three family members (Aurora A, B, and C) have been identified independently, and the nomenclature designates the original Drosophila Aurora kinase as Aurora A (Carmena and Earnshaw, Nat. Rev. Mol. Cell. Biol., 2003, 4, 842-854).
Using a PCR-based method to screen for novel serine/threonine kinases, the cDNAs encoding mouse and human Aurora B (also known as serine/threonine kinase 12 AIK2; AIM-1; AIM1; AIRK-2; ARK2; Aurora/IPL1-like kinase 2; HsAurora-1; STK12) were isolated. The Aurora B protein contains a kinase domain with high homology to the catalytic domain of other serine/threonine kinases (Shindo et al., Biochem. Biophys. Res. Commun., 1998, 244, 285-292). A kinase-negative mutant was shown to induce a failure in cytokinesis during mitosis, indicting mammalian Aurora B in mitotic processes (Terada et al., EMBO J., 1998, 17, 667-676). The Aurora B gene maps to chromosome 17p13, a region that is deleted in various cancers (Giet and Prigent, J. Cell Sci., 1999, 112 (Pt 21), 3591-3601), however high levels of expression are found in cell lines from colorectal tumors (Tatsuka et al., Cancer Res., 1998, 58, 4811-4816), making Aurora B an attractive target for anti-cancer therapies.
Subsequent studies have shown that aurora B kinases form a complex with at least two other proteins, the inner centromere protein (INCENP) and survivin. These proteins behave as “chromosomal passenger” proteins, first localizing along the chromosomes during prophase before concentrating at the centromere during prometaphase. During anaphase, the complex relocates to the central spindle. It is thought that the active component in the passenger complex is the Aurora B kinase, and indeed, Aurora B can phosphorylate survivin in vitro at a site important for the in vivo localization of survivin in human cells (Wheatley et al., J. Biol. Chem., 2004, 279, 5655-5660). However, survivin has been shown to enhance Aurora B kinase activity and to localize Aurora B to its substrates during the cell cycle (Chen et al., J. Biol. Chem., 2003, 278, 486-490). Likewise, INCEP is both a substrate and an enhancer of the kinase (Carmena and Earnshaw, Nat. Rev. Mol. Cell. Biol., 2003, 4, 842-854; Honda et al., Mol. Biol. Cell, 2003, 14, 3325-3341). The phase-dependent localization of the complex coupled with Aurora B functional studies suggest that the protein is involved in chromosome structure and alignment as well as in coordinating anaphase and cytokinesis (Adams et al., Trends Cell Biol., 2001, 11, 49-54; Shannon and Salmon, Curr. Biol., 2002, 12, R458-460).
Several studies have further confirmed the importance of mammalian Aurora B in several different stages of cell division. Expression of a dominant negative mutant of Aurora B lacking kinase activity in mammalian NRK tissue cells caused multiple defects in the mitotic machinery. Notably, centromere movement was altered, and kinetochores became separated from spindle microtubles. Levels of the motor proteins dynein and CENP-E were diminished at prometaphase kinetochores, suggesting that Aurora B kinase activity plays a major role in the kinetochore assembly pathway (Murata-Hori and Wang, Curr. Biol., 2002, 12, 894-899). Antisense DNA depletion of mitotic centromere-associated kinesin (MCAK), another Aurora B substrate, in mammalian cells has been shown to inhibit anaphase A. Disruption of Aurora B kinase function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK, while Aurora B phosphorylation of MCAK actually inhibits its microtubule depolymerizing activity in vitro, an action which may prevent reversal of the initial microtubule-kinetochore attachments (Andrews et al., Dev. Cell, 2004, 6, 253-268). CENP-A, a histone H3 variant and kinetochore protein, is a known target of Aurora B. However, mutations affecting the phosphorylation of this protein do not disrupt mitosis, but do prevent completion of cytokinesis (Zeitlin et al., J. Cell. Biol., 2001, 155, 1147-1157).
The two sister kinetochores on each chromosome must become attached to opposing spindle poles for proper segregation of replicated chromatids. If a kinetochore is unattached under normal conditions, anaphase is suppressed by the spindle assembly checkpoint. If both sister kinetochores become attached to the same pole, the monoorientation would result in improper segregation. Studies with hesperadin, a small molecule inhibitor of Aurora B, suggest that the kinase is required to generate unattached kinetochores on monooriented chromosomes, thus promoting bipolar attachment and maintaining checkpoint signalling (Hauf et al., J. Cell Biol., 2003, 161, 281-294). Exploiting the reversibility of small-molecule inhibitors of Aurora kinase activity, hesperadin and Aurora kinase inhibitor-1, it was demonstrated that controlled activation of Aurora kinases resulted in correction of chromosome attachment errors by selective disassembly of kinetochore-microtubule fibers (Lampson et al., Nat. Cell Biol., 2004, 6, 232-237). Thus Aurora B functions to maintain genome integrity during cell division not only by coordinating kinetochore assembly, but also by correcting attachment errors thus enabling the spindle assembly checkpoint (Hauf et al., J. Cell Biol., 2003, 161, 281-294; Lampson et al., Nat. Cell Biol., 2004, 6, 232-237).
The myriad roles of Aurora B in cell division make it an attractive target for therapeutic and investigative strategies aimed at disorders of cell proliferation, including hyperproliferative disorders. Consequently, there remains a need for agents capable of effectively modulating Aurora B function.
Antisense technology is an effective means for reducing the expression of specific gene products and is therefore uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of Aurora B expression.
The present invention provides compositions and methods for modulating Aurora B expression.